Nucleic acids are commonly amplified in diagnostics, research, and forensics to preserve samples, identify pathogens and clone DNA. A variety of techniques have been developed to amplify nucleic acids. Amongst these, the polymerase chain reaction (PCR) is the most widely known. PCR, a target amplification technique (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159), utilizes two oligonucleotides primers that hybridize to the 5′ and 3′ edges of a target sequence. A DNA polymerase extends the primers through the addition of deoxynucleoside-triphosphates (dNTPs) to create double-stranded product. The double-stranded products are separated through the use of heat and the process is repeated to generate exponential amplification of the selected target.
In addition to PCR, a variety of other methods of nucleic acid amplification have been developed. Examples of these are strand displacement amplification (SDA), which utilizes restriction enzyme nicking of a DNA strand followed by extension with an exonuclease-deficient polymerase (U.S. Pat. Nos. 5,455,166 and 5,470,723), transcription mediated amplification (TMA), which utilizes an RNA polymerase to generate an RNA copy from the target sequence, combined with a reverse transcriptase to generate DNA (U.S. Pat. Nos. 5,399,491 and 5,554,516), and ligase chain reaction (LCR), a signal amplification method (U.S. Pat. No. 5,494,810). In addition, a method has been developed that utilizes a helicase to perform in vitro strand separation for the purposes of amplification of DNA or RNA (U.S. Pat. No. 7,282,328). This method, entitled Helicase Dependent Amplification (HDA), has performance capabilities similar to PCR in an isothermal format. HDA can be performed at many different temperatures, dependent upon the polymerase and helicase selected for the assay.
The detection of amplified nucleic acids for clinical applications primarily relies upon nucleic acid hybridization to sequence-specific probes. This ensures that the product generated is the intended amplified sequence and not a spurious artifact or primer dimer. Asymmetric PCR is a method well known in the art (U.S. Pat. No. 5,066,584) and was developed to produce increased quantities of single-stranded amplicon from PCR-based amplification required for use in direct sequencing reactions and/or probe hybridization.
The hybridized probe can be visualized through a variety of methods well known in the art that include the labeling of the probe with enzymes or luminescent or fluorescent reagents. The use of probes comprised of oligonucleotide sequences bound to microparticles is also well known in the art. The detection of an amplified product with luminescent or fluorescently labeled probes requires the use of specialized equipment such as real-time thermocyclers or luminometers. Fluorescent detection of amplified nucleic acids is a rapid and sensitive method, but it significantly increases the cost of nucleic acid diagnostics. An alternative method to detect the amplified nucleic acid products is through the use of a membrane-based test strip similar to the lateral-flow immunoassays (or “dipsticks”) widely used in protein detection. Lateral-flow detection is advantageous over machine-based methods in that the method can be performed with small, inexpensive and disposable devices (see, for example, U.S. Pat. Nos. 5,955,351 and 5,344,757 and US Patent Application Publication Nos. 2006/0160078 A1 and 2009/0181388 A1. Methods of lateral-flow test strip detection are well known in the art, primarily as a means to detect proteins, known as lateral-flow immunoassays. For example, non-radioactively labeled molecules such as biotin can be attached to oligonucleotide probes and captured on a solid phase support membrane for detection. Furthermore, capture may be mediated through an antibody-antigen reaction, wherein an antibody incorporated into the test strip to a hapten conjugated to the oligonucleotide is captured and visualized through dye-particles that generate a visible line. This allows for the specific detection of the amplified product on a lateral-flow test strip without any instrumentation (see, for example, US Patent Application Publication No. 2009/0181388 A1 and Goldmeyer et al., J. Clin. Microbiol. 46: 1534-1536 (2008)).
In nucleic acid amplification tests (NAAT), inhibitors of amplification reactions can be present in clinical samples. Inhibition of amplification can result in false negatives whereby the inability to detect the target of interest is caused by the failure of the reaction rather than the lack of target present. A false negative can result in the misdiagnosis of a disease or infection. To detect inhibition that may prevent amplification, resulting in a false negative, the use of an internal amplification control is necessary. This concept has been described for use in amplification reactions performed and monitored fluorescently in real-time thermocyclers. A variety of methods have been previously described which detail the use of internal controls for real-time amplification (see, for example, U.S. Pat. No. 6,312,929 and US Patent Application Publication Nos. 2005/0003374 A1 and 2006/0166232 A1). One internal control design, called a “competitive internal control,” utilizes a single pair of primers to simultaneously amplify both a target sequence of interest and a reference “internal control” sequence that can be amplified even when the target sequence is undetectable (J. Clin. Microbiol. 32:1354-1356 (1994)). However, the use of an internal control in conjunction with the detection of enzymatically amplified nucleic acids on a lateral-flow strip and the methods to do so have not been previously described.